Characterization of the complete chloroplast genome of the rare medicinal plant: Mandragora caulescens (Solanaceae)

Abstract In this study, we assembled high-quality chloroplast genomes of Mandragora caulescens through a reference-guided approach using high-throughput Illumina sequencing reads. The resulting chloroplast genome assembly displayed a typical quadripartite structural organization, comprising a large single-copy (LSC) region of 85,233 bp, two inverted repeat (IR) regions of 25,685 bp each, and a small single-copy (SSC) region of 18,207 bp. The chloroplast genome harbored 141 complete genes, and its overall GC content was 38.0%. In maximum-likelihood (ML) and Bayesian inference (BI) trees, the 19 Solanaceae species formed a monophyletic group, dividing into two main clades. M. caulescens and Nicandra physalodes formed a monophyletic group, suggesting a close relationship between the two species. The M. caulescens cp genome presented in this study lays a good foundation for further genetic and genomic studies of the Solanaceae.


Introduction
Mandragora caulescens C. B. Clarke (Clarke 1883), a rare medicinal plant, belongs to the tribe Solaneae (Solanaceae).It mainly grows in the meadows and alpine meadows (2200-4200 m a.s.l.) in western Sichuan, northwestern Yunnan, and eastern Tibet of China (Zhang et al. 1994;Mabberley 2017).The roots of this plant contain high levels of total alkaloids, with the main components being anisodamine and apoatropine (Wang et al. 2002;Wan et al. 2011).Previous research has indicated that both the roots and aerial parts of the plant are commonly used in traditional Chinese medicine, renowned for their ability to alleviate pain and treat certain skin conditions (Maity et al. 2019).
The chloroplast is a semi-autonomous eukaryotic organelle that has a small genome (cp genome) that interacts with the nuclear and mitochondrion genome to provide the biochemical machinery for energy conversion (Li et al. 2015;Hollingsworth et al. 2016).Recent utilization of the complete chloroplast genome in addressing phylogenetic issues has been increasing, attributed to its distinct characteristics and its ability to reveal valuable information worth considering (Guo et al. 2017;Zhang et al. 2018;Xie et al. 2022;Ran et al. 2024).The complete chloroplast genomes have been extensively employed in resolving some lingering queries in plant taxonomy recently (Hu et al. 2016;Yang et al. 2022).Besides the evolutionary aspect, the chloroplast genome holds significant implications in chloroplast transformation (Daniell et al. 2008).A comprehensive examination of Mandragora caulescens could have notable implications for comprehending the origin and evolution of the Solanaceae.Previous phylogenetic investigations of M. caulescens primarily relied on a limited number of chloroplast genes or ITS (Volis et al. 2015(Volis et al. , 2018)), Nevertheless, the chloroplast genome of Mandragora caulescens remains unreported.
In this study, we assembled and analyzed the complete chloroplast genome of M. caulescens for the first time.

Materials
Fresh leaves of florescence of M. caulescens were gathered from Balang mountains (Wenchuan, Sichuan, China; coordinates: 102.9147E, 30.8922N) (by Lei Zhang: zhangsanshi-0319@163.com) (Figure 1(A)), and desiccated using silica gel.The voucher specimen was archived in the Herbarium of North Minzu University with an accession number of zlnmu2023116 (Figure 1(B)).0.5 g of the dry leaf sample was utilized for DNA extraction.

Methods
Total genomic DNA was isolated with a modified CTAB method (Doyle and Doyle 1987) Kit was employed to create the sequencing libraries following the manufacturer's instructions.DNA was randomly sheared to a size of 350 bp.This library was sequenced on the Illumina NovaSeq 6000 platform with 150 bp paired-end read length.We acquired 6.2 Gb of high-quality paired-end reads for M. caulescens.After eliminating the adapters, two approaches were used to assemble the cp genome, we de novo assembled the cp genome M. caulescens using NOVOPlasty 4.1 (Dierckxsens et al. 2017) with the specified parameters: k-mer ¼ 39 and genome range 120,000-200,000 bp.The other approach was assembled using GetOrganelle (Jin et al. 2020) (Frazer et al. 2004) and the annotated CPG of Capsicum annuum was used as the reference in the LAGAN mode (Brudno et al. 2003).

Discussion and conclusions
Previous research has indicated that land plant cp genomes typically range from 120 to 160 kb (Wicke et al. 2011).In this investigation, in this study, the complete cp genome of M. caulescens was assembled for the first time, and the structure of this species was annotated.It exhibited a standard quadripartite structure with a total sequence length of 154,810 bp, encompassing the LSC region (85,233 bp), the SSC region (18,207 bp), and two identical IR regions (25,685 bp).The GC content of M. caulescens plants is 38.0%, consistent with findings from various angiosperm studies where the highest GC content was observed in the IR regions (Wu et al. 2020).
The cp genome serves as a focal point in molecular biology research and has emerged as a current area of interest for species genealogy identification (Ran et al. 2024).In this study, a phylogenetic tree was developed utilizing the BI method and the ML method.The tree revealed that the cp the cp genomes of Solanaceae species clustered together with strong support, with M. caulescens and Nicandra physalodes forming a monophyletic group.This phylogenetic result was consistent with Volis et al. (2018).The alignments indicated high sequence similarity among the CPGs of the 19 Solanaceae species (Supplementary Fig. S6).However, sequence divergence in non-coding regions was greater than that in coding regions.Further investigation of M. caulescens is necessary, including additional studies at the population level and genome analysis, as well as examining the distribution of the population.Only through thorough analysis can the adaptive differentiation of M. caulescens be understood.In addition, expanding the collection of Solanaceae cp genomes will provide deeper insights into the evolution of this ecologically and economically important plant family.

ORCID
Yuqing Wei http://orcid.org/0000-0002-9106-774XLei Zhang http://orcid.org/0000-0001-5301-4658 . Sequence lengths obtained by the two methods are similar.The complete chloroplast genome sequence of Physalis angulata (MH045574) was utilized as a reference.Plann v1.1(Huang and Cronk 2015) was employed for annotating the chloroplast genome, and Geneious v11.0.3(Kearse et al. 2012) was used for correcting the annotation.The sequencing depth coverage was conducted by Samtools(Li et al. 2009).To further elucidate the phylogenetic placement of M. caulescens in Solanaceae, the chloroplast genomes of 19 representative species were retrieved from NCBI GenBank to reconstruct the chloroplast genome phylogenetic tree, with Convolvulus arvensis serving as an outgroup.Protein-coding genes (PCGs) were extracted from the GenBank formatted file containing 20 plastomes using customized Perl scripts, removing start and end codons.A total of 56 PCGs were retained for all species.Each PCG was aligned using PRANK v.130410 (L€ oytynoja and Goldman 2008) according to the translated amino acid sequences.Phylogenetic analyses were performed for each dataset (CDS and WP) using both maximum-likelihood (ML) and Bayesian inference (BI) strategies.We used RAxML v8.1.24(Stamatakis 2014) to conduct ML analyses with the GTR þ C model.The optimal model (GTR þ I þ G) was identified using jModeltest and BI analysis was conducted in MrBayes v 3.2.6(Ronquist et al. 2012).FigTree v1.4.2(Rambaut 2012) was subsequently utilized to visualize the phylogeny.The results of the comparative analysis of the CPGs were visualized with the mVISTA program

Figure 2 .
Figure 2. The detailed genome map of M. caulescens cp genome.The large single-copy (LSC), small single-copy (SSC) region and two inverted repeat regions (IRA and IRB), and GC content (light gray) are shown in the inside track.Gene models including protein-coding genes, tRNA genes and rRNA genes are shown with various colored boxes in the outer track.
. The NEBNext DNA Library Ecotones in the Yellow River Basin National Ethnic Affairs Commission of the People's Republic of China, North Minzu University, Yinchuan, Ningxia, PR China Supplemental data for this article can be accessed online at https://doi.org/10.1080/23802359.2024.2368213.